The plasminogen activator (PA) system involves the serine proteases plasmin and urokinase plasminogen activator (uPA); the serpins α2-antiplasmin, plasminogen activator inhibitor type-1 (PAI-1) and plasminogen activator inhibitor type-2 (PAI-2); and the uPA receptor (uPAR). During the past decade, evidence for the involvement of components of the PA system in cancer metastasis has increased and it is believed that the uPA-mediated pathway of plasminogen activation is active in the cancer process. (P. A. Andreasen et al., 1997, Int. J. Cancer, 72:1-22).
Proteolytic enzymes, such as those of the PA system, are involved in cancer invasion and metastasis by virtue of their ability to invade and degrade basement membranes and extracellular matrix proteins that surround normal tissue (J. H. DeWitte et al., 1999, Br. J. Cancer, 79:1190-1198; L. A. Liotta et al., 1982 Cancer Metastasis Rev., 1:277-297; K. Dano et al., 1985, Adv. Cancer Res., 44:139-266; P. Mignatti and D. B. Rifkin, 1993, Physiol. Rev., 73:161-195; and P. A. Andreasen et al., 1997, Ibid.). Immunohistochemical and in situ observations of uPA, plasminogen and PAI-1 distribution in adenocarcinomas show that proteinase degradation of the extracellular matrix occurs as localized invasive foci (J. Grøndahl-Hansen et al., 1991, Am. J. Pathol., 138:111-117; C. Pyke et al., 1991, Proc. Third Intl. Workshop on the Molecular and Cellular Biology of Plasminogen Activation:Elsinore, 45; C. Pyke et al., 1991, Cancer Res., 51:4067-4071). In the case of angiogenesis, there is also a functional interaction between uPA and PAI-1 (E. Bacharach et al., 1992, Proc. Natl. Acad. Sci. USA, 89:10686-10690).
Urokinase plasminogen activator (uPA) is a 52 kilodalton (kDa) serine protease that is secreted by cells as an inactive, single-chain precursor called pro-uPA. Enzymatic cleavage of pro-uPA at lysine 158 produces an active heterodimer, called high molecular weight uPA (HMW-uPA), which contains two subunits A and B. When pro-uPA is secreted from cells, it binds to uPAR on the cell surface through an EGF-like domain on the A chain. Subsequent binding of plasmin to uPA can convert pro-uPA into the proteolytically active heterodimer. In turn, active uPA rapidly converts the inactive plasmin precursor, plasminogen, into enzymatically active plasmin, which is directly involved in extracellular matrix degradation, as well as in the activation of other pro-collagenases, some prometalloproteases and latent growth factors (K. Dano et al., 1985, Ibid.; M. J. Duffy, 1992, Clin. Exp. Metastasis, 10:145-155; J. R. Pollanen et al., 1991, Adv. Cancer Res., 57:273-282; L. Ossowski, 1992, Cancer Res., 52:D:6754-6760; P. Mignatti and D. B. Rifkin, 1993, Ibid.; and P. A. Andreasen et al., 1997, Ibid.). The additional cleavage of uPA after lysine 135 releases the 17 kDa amino terminal fragment (ATF), leaving the carboxy-terminal low molecular weight uPA (LMW-uPA, 33 kDa), which retains full catalytic activity. (F. Blasi et al., 1990, Seminars in Cancer Biology, 1:117-126).
Both PAI-1 and PAI-2 bind to the catalytically active B chain of uPA to regulate its enzymatic activity. By forming complexes with uPA bound to uPAR on the cell surface, PAI-1 promotes the clearance of proteolytic activities from the cell surfaces, as well as the recycling of unbound uPAR back to the cell surface, thereby regulating the overall invasive and metastatic behavior of cancer cells. (P. A. Andreasen et al., 1997, Ibid. and H. A. Chapman et al., 1997, Curr. Op. Cell Biol., 9:714-724). PAI-1 is a 50 kDa glycoprotein serine protease inhibitor that is the principal physiological inhibitor of both forms of the plasminogen activators PA and tissue plasminogen activator (TPA). PAI-1 is secreted in an active form which spontaneously converts to a latent form (G. Deng et al., 1995, Thrombosis and Haemostasis, 74:66-70), but it can be stabilized in the active form by binding to the plasma protein vitronectin (D. A. Lawrence et al., 1994, J. Biol. Chem., 269:15223-15228). Both tumor cells and capillary endothelial cells express higher levels of PAI-1 than do other cell types (K. Bajou et al., 1998, Nature Medicine, 4:923-928). High levels of PAI-1 are thought to protect the tumor stroma from degradation by the high amounts of uPA secreted by cells. (E. Bacharach et al., 1992, Proc. Natl. Acad. Sci. USA, 89:10686-10690; P. Kristensen et al., 1990, Histochemistry, 93:559-566). Elevated levels of PAI-1 may also contribute to tumor-induced angiogenesis by protecting the extracellular matrix surrounding the tumor from proteolytic degradation (C. Pyke et al., 1991, Cancer Res., 51:4067-4071). When active uPA is bound to its receptor, the subsequent binding of PAI-1 results in internalization and degradation of the uPA:uPAR:PAI-1 complex. (M. V. Cubellis et al., 1990, The EMBO J., 9:1079-1085). This down-regulation of uPA decreases the amount of active uPA on the cell surface.
Secreted uPA can originate from several cell types, including tumor cells (G. Markus et al., 1983, Cancer Res., 43:5517-5525), adjacent stromal cells and fibroblasts (C. Pyke et al., 1991, Am. J. Pathol., 138:1059-1067). Early studies of PAI-1 and uPA:PAI-1 complexes in oncogenesis involved the use of tumor lysates and cytosols; it was found that PAI-1 levels in tumor lysates had a prognostic correlation in breast cancer. Tumor levels of PAI-1 were also analyzed in lung cancer, colon cancer and renal cell carcinoma; this inhibitor has become an unlikely prognostic marker in tumor tissue for cancer metastasis. (P. A. Andreasen et al., 1997, Ibid.).
Because the PA system components are intricately involved in the process of cancer and cancer spread in a variety of cancer types, which afflict both genders, it is a problem in the art to be able to accurately and sensitively screen over time to determine and monitor those individuals who are likely to respond, and/or who are responding to, (or not responding to), or benefiting from (or not benefiting from), anti-cancer therapy(ies), or combination therapies, particularly, molecularly targeted therapies to the plasminogen activation system. The present invention solves such a problem by providing a sensitive and reliable assay method, preferably an immunoassay, to determine levels of PA system analyte components in body fluid samples of cancer patients compared to the levels of these respective PA system components in normal individuals. In addition, the present invention is advantageous in that it is employed to monitor cancer patients undergoing cancer or anti-neoplastic therapies to treat cancers associated with the activity of PA system components to assist in the determination and examination of cancer treatment regimens and patient progress and/or outcome during the course of disease and/or anti-cancer therapy(ies).